This program of research comprises an integrated study of cellular and molecular processes that regulate contraction and kinetics of contraction in mammalian myocardium. Objectives are to determine (1) mechanisms of Ca2+ delivery during excitation-contraction coupling, (ii) mechanisms of regulation of mechanical properties, and (iii) mechanisms of action of the PKA and PKC signaling pathways. The program is composed of five sub-projects and four cores. Dr. Kamp's project will study protein kinase C (PKC) regulation of Ca2+ entry into the myocardium, focusing on the modulation of L-type Ca2+ channels and the specific role of phosphorylation of the DHP receptor and the ryanodine receptor and will investigate the molecular basis for differences in excitation-contraction coupling between skeletal and cardiac muscles. Dr. Valdivia's project will investigate the emphasis on the factors and protein components of the Ca2+ release unit that are responsible for shutting off Ca2+ release during excitation-contraction coupling. Sub-project 4 will examine mechanisms of regulation of mechanical properties of single skinned myocytes, and will emphasize gene knock-out, selective extraction, and mutation of myofibrillar proteins thought to mediate or modulate regulation. Sub- project 5 will investigate the molecular mechanisms of regulation of cardiac contraction by protein kinase C, with emphasis on effects involving Ca2+, entry, Ca2+ release from the sarcoplasmic reticulum, and contractile protein interaction. Scientific cores focused on molecular biology and gene targeting, lipid and peptide probes of cellular processes, and production of differentiated cardiac myocytes from embryonic stem cells will provide support to the sub-projects and will facilitate development of new research directions. Our uniquely complementary approaches will yield new information concerning the subcellular processes that mediate excitation-contraction coupling and regulate myocyte function in the heart.